Dissociation of hemoglobin into subunits. Ligand-linked dissociation at neutral pH

The dimer-tetramer association-dissociation equilibrium of hemoglobin is strongly ligand-linked at neutral pH; the ratio of the association constant of unliganded to that of oxyhemoglobin is estimated from ultracentrifuge data to be not less than 10³ in sodium chloride solutions and probably not less than 10⁵ in sodium iodide solutions. This finding affords an explanation of the fact that the ligand binding characteristics of hemoglobin are to a first approximation independent of the degree of dissociation of oxyhemoglobin—the “salt paradox”.     

Dissociation of aspartate aminotransferase into subunits. Effect of ligands upon this dissociation.

Frontal and zonal analysis of the chromatography of aspartate aminotransferase (EC2.61.1), pig heart cytosolic enzyme, on Bio-Gel P150 shows that holo- and apoenzyme can dissociate at pH 8.3. Ultracentrifugation and fluorescence depolarization confirm this result. Kinetic analysis of the fluorescence depolarization experiments favors a biphasic phenomenon: a few minutes for the faster one and several hours for the slower one. The apparent dissociation constant is 0.8 muM for the apoenzyme and 0.18 muM for the pyridoxal 5'-phosphate form of the holoenzyme. In the presence of sucrose or 0.1 M L-aspartate or a mixture of 70 mM L-glutamate and 2 mM alpha-ketoglutarate, the holoenzyme is dimeric at concentrations higher than 5 nM. The addition of a mixture of the substrates L-glutamate and alpha-ketoglutarate to a monomeric holoenzyme leads to dimerization. The stability of the dimeric form is in the order: holoenzyme + substrates greater than apoenzyme.     

Monomer diffusion into polymer domains in sickle hemoglobin.

The gelation of sickle hemoglobin includes the formation of spherulitic arrays of polymers, known as polymer domains, which are an intrinsic result of the polymer formation mechanism. We have observed the diffusion of monomers into domains as they form, which substantially increases the total concentration of hemoglobin within the domain. The maximum total concentration attained is comparable with the pellet concentration of 0.5-0.55 g/cm3 obtained in sedimentation experiments. The half time for this process is approximately 50 s for domains of 25 microns radius, and is approximately independent of temperature. The shape of the diffusion progress curves as well as the deduced diffusion constants, and their weak temperature dependence are consistent with a simple model of hemoglobin monomer diffusion into the domain.     

Dissociation of the ribulosebisphosphate-carboxylase large-subunit binding protein into dissimilar subunits

The ribulosebisphosphate-carboxylase large-subunit binding protein from Pisum sativum chloroplasts is an oligomer of two types of subunit with the composition alpha 6 beta 6. These two subunits are immunologically distinct, show different partial protease digestion patterns and have different amino-terminal sequences. Leaves of Hordeum vulgare also contain an oligomeric binding protein composed of equal amounts of two types of subunit. Treatment of either P. sativum stromal extracts or purified binding protein with ATP and Mg2+ ions causes the dissociation of the oligomeric form of the binding protein to the monomeric subunits. This effect is highly specific for ATP since CTP, UTP, GTP, ADP, AMP, cyclic AMP, NADPH and pyrophosphate do not cause dissociation.     

Dissociation of small-intestinal sucrase - isomaltase complex into enzymatically active subunits.

1. The sucrase - isomaltase complex from rabbit small intestine dissociated into its subunits upon reaction with citraconic anhydride. They can recombine after deacylation under mild acidic conditions. 2. When citraconylated, the subunits could be separated and isolated in a catalytically active form. 3. The previously reported procedure for separation of the subunits by alkaline treatment at pH 9.6 is apparently not due to contaminating degradative enzymes (possibly still present at undetectable levels in the isolated sucrase - isomaltase complex) but to the action of alkali.     

Dissociation of mitochondrial malate dehydrogenase into active soluble subunits

Gel exclusion chromatographic studies demonstrate that cytosolic and mitochondrial malate dehydrogenases (cMDH and mMDH) dissociate into subunits in the presence of 0.1% of the non-ionic detergent Triton X-100 (TX-100). The presence of cofactor and catalytically competent cofactor-substrate pairs does not protect mMDH against this dissociation. In contrast, cMDH dimers resist dissociation in the presence of either addition. Since steady state kinetic studies indicate both enzymes are fully active in the presence of 0.1% TX-100, we conclude that quaternary structure is not a kinetically important feature of mMDH structure and cooperativity does not account for mMDH kinetic anomalies. In contrast, cooperativity is a reasonable explanation for cMDH kinetic properties even in the presence of 0.1% TX-100, since this enzyme's subunits associate in the presence of active site ligands. The existence of fully active mMDH subunits raises the possibility that this species rather than the dimer may be a constituent of proposed multi-enzyme complexes of the mitochondrion. Preliminary chromatographic experiments involving gently disrupted mitochondria have found MDH activity in differently sized complexes, all with molecular weights larger than the mMDH dimer but smaller than complexes anticipated for multi-enzyme complexes involving other enzymes and the mMDH dimer.     

The effect of hemoglobin ligands on the kinetics of human hemoglobin A1c formation

HbA1c is the most prevalent of the minor human hemoglobins. It is formed by the nonenzymatic addition of glucose to the alpha-amino group of the beta chain by an initial condensation reaction and a subsequent intermolecular Amadori rearrangement. We have developed a method of analysis which utilizes high performance liquid chromatography to follow the formation of HbA1c and greatly simplifies the determination of the kinetic parameters associated with this reaction. This has allowed us to study the effects of several Hb ligands, including the hydrogen ion, on the kinetics of this glycosylation reaction. Both the initial condensation reaction and the subsequent rearrangement are shown to exhibit acid catalysis, but the rate of the condensation step is limited by the extent of protonation of the alpha-amino group. The variation in kinetic parameters as a function of hydrogen ion concentration has allowed us to determine the probable reaction mechanism of HbA1c formation by comparison to previously reported model systems of Schiff base formation and Amadori rearrangement. The formation of pre-HbA1c from deoxy-Hb shows an increased forward rate when compared to oxy-Hb. The presence of physiologic concentrations of CO2 causes a proportional decrease in both k1 and k-1. 2,3-Diphosphoglycerate causes a significant increase in the keq of the formation reaction. The effects of CO and the substitution of L-glucose for D-glucose are not significant.